Self-Quenched Fluorophore-DNA Labels for Super-Resolution Fluorescence Microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Protein labeling with fluorophore-DNA oligonucleotides is key for super-resolution microscopy.
• Current methods face challenges with high background fluorescence from unbound labels.
• Multitarget imaging and molecular quantification are crucial applications.

Purpose of the Study:

• To introduce a novel concept of self-quenching fluorophore dimers conjugated to DNA oligonucleotides.
• To reduce background fluorescence in optical super-resolution microscopy.
• To enhance multitarget imaging and molecular quantification capabilities.

Main Methods:

• Conjugation of fluorophore dimers to DNA oligonucleotides for self-quenching.
• Characterization of fluorophore spectra and hybridization equilibria.
• Application of self-quenched labels in stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM).

Main Results:

• Demonstrated effective reduction of background fluorescence through self-quenching.
• Observed restoration of fluorescence signals upon specific hybridization.
• Expanded the range of suitable fluorophores for self-quenching applications.
• Reported improved imaging performances in STED and SMLM.

Conclusions:

• Self-quenching fluorophore-DNA labels offer a general strategy to minimize background noise.
• This approach significantly enhances the quality of super-resolution microscopy imaging.
• The developed method improves multitarget imaging and molecular quantification accuracy.